MiR-29b-1-5p is altered in BRCA1 mutant tumours and is a biomarker in basal-like breast cancer.

Depletion of BRCA1 protein in mouse mammary glands results in defects in lactational development and increased susceptibility to mammary cancer. Extensive work has focussed on the role of BRCA1 in the normal breast and in the development of breast cancer, the cell of origin for BRCA1 tumours and the protein-coding genes altered in BRCA1 deficient cells. However, the role of non-coding RNAs in BRCA1-deficient cells is poorly understood. To evaluate miRNA expression in BRCA1 deficient mammary cells, RNA sequencing was performed on the mammary glands of Brca1 knockout mice. We identified 140 differentially expressed miRNAs, 9 of which were also differentially expressed in human BRCA1 breast tumours or familial non-BRCA1 patients and during normal gland development. We show that BRCA1 binds to putative cis-elements in promoter regions of the miRNAs with the potential to regulate their expression, and that four miRNAs (miR-29b-1-5p, miR-664, miR-16-2 and miR-744) significantly stratified the overall survival of basal-like tumours. Importantly the prognostic value of miR-29b-1-5p was higher in significance than several commonly used clinical biomarkers. These results emphasise the role of Brca1 in modulating expression of miRNAs and highlights the potential for BRCA1 regulated miRNAs to be informative biomarkers associated with BRCA1 loss and survival in breast cancer.

Identification of fifteen novel germline variants in the BRCA1 3'UTR reveals a variant in a breast cancer case that introduces a functional miR-103 target site.

Mutations in the BRCA1 gene confer a substantial increase in breast cancer risk, yet routine clinical genetic screening is limited to the coding regions and intron-exon boundaries, precluding the identification of mutations in noncoding and untranslated regions (UTR). As 3'UTR mutations can influence cancer susceptibility by altering protein and microRNA (miRNA) binding regions, we screened the BRCA1 3'UTR for mutations in a large series of BRCA-mutation negative, population and clinic-based breast cancer cases, and controls. Fifteen novel BRCA1 3'UTR variants were identified, the majority of which were unique to either cases or controls. Using luciferase reporter assays, three variants found in cases, c.* 528G>C, c.* 718A>G, and c.* 1271T>C and four found in controls, c.* 309T>C, c.* 379G>A, c.* 823C>T, and c.* 264C>T, reduced 3'UTR activity (P < 0.02), whereas two variants found in cases, c.* 291C>T and c.* 1139G>T, increased 3'UTR activity (P < 0.01). Three case variants, c.* 718A>G, c.* 800T>C, and c.* 1340_1342delTGT, were predicted to create new miRNA binding sites and c.* 1340_1342delTGT caused a reduction (25%, P = 0.0007) in 3'UTR reporter activity when coexpressed with the predicted targeting miRNA, miR-103. This is the most comprehensive identification and analysis of BRCA1 3'UTR variants published to date.

Effect of BRCA2 sequence variants predicted to disrupt exonic splice enhancers on BRCA2 transcripts.

BACKGROUND: Genetic screening of breast cancer patients and their families have identified a number of variants of unknown clinical significance in the breast cancer susceptibility genes, BRCA1 and BRCA2. Evaluation of such unclassified variants may be assisted by web-based bioinformatic prediction tools, although accurate prediction of aberrant splicing by unclassified variants affecting exonic splice enhancers (ESEs) remains a challenge. METHODS: This study used a combination of RT-PCR analysis and splicing reporter minigene assays to assess five unclassified variants in the BRCA2 gene that we had previously predicted to disrupt an ESE using bioinformatic approaches. RESULTS: Analysis of BRCA2 c.8308 G > A (p.Ala2770Thr) by mRNA analysis, and BRCA2 c.8962A > G (p.Ser2988Gly), BRCA2 c.8972G > A (p.Arg2991His), BRCA2 c.9172A > G (p.Ser3058Gly), and BRCA2 c.9213G > T (p.Glu3071Asp) by a minigene assay, revealed no evidence for aberrant splicing. CONCLUSIONS: These results illustrate the need for improved methods for predicting functional ESEs and the potential consequences of sequence variants contained therein.

Targeted disruption of Brca1 in restricted compartments of the mouse mammary epithelia.

Tumours arising in BRCA1 mutation carriers have a characteristic phenotype, the molecular and cellular basis of which is unknown. To address the hypothesis that this phenotype reflects a role for BRCA1 in either in the basal or the stem cell compartments of the mammary epithelia, we have targeted its disruption to K14 and K6a expressing cells of the mouse. Unlike MMTV and WAP driven conditional knockout models of Brca1, these two models did not result in any observable changes in the mammary gland. Our results suggest that BRCA1-associated tumours arise either in K14 and K6a negative basal cells of the mammary gland, or possibly from transdifferentiation of luminal epithelia.